PROJECT SUMMARY Skeletal muscle is a unique organ that is composed of multinucleate muscle fibers, each of which is the product of fusion of hundreds or even thousands of myoblasts. Myoblast fusion is not only important for skeletal muscle development, but also critical for satellite cell-based muscle regeneration. Despite a large body of studies over several decades, the mechanisms underlying myoblast fusion in humans remain poorly understood. Recent studies in the fruit fly Drosophila have begun to reveal unprecedented details about the molecular and cellular mechanisms of myoblast fusion. The striking evolutionary conservation between fly and mammalian myogenesis makes Drosophila a particularly relevant system to study myoblast fusion in vivo. Recent studies from our lab have uncovered a novel cellular mechanism underlying myoblast fusion. We show that myoblast fusion is mediated by F-actin-enriched podosome-like structure (PLS), which invades the apposing fusion partner with multiple protrusive fingers leading to fusion pore formation. Furthermore, we have discovered a mechanosensory response in the receiving fusion partner and demonstrated that mechanical tension is a driving force for myoblast fusion. In this proposal, we will build upon our previous work and expand our studies into two new directions ? calcium signaling and phospholipid function in myoblast fusion. We will use a multifaceted approach, including fly genetics, molecular biology, biochemistry, immunohistochemistry, live imaging, super-resolution microscopy and electron microscopy, to study the mechanisms by which calcium and PIP2 signaling regulate myoblast fusion. Given the molecular and cellular conservation of myoblast fusion between fly and mammals, the proposed mechanistic studies using the simpler and genetically tractable Drosophila system will lead to significant insights into human muscle biology in health and disease, and ultimately provide a basis for developing more efficient therapeutics against the life debilitating muscle degeneration diseases.